Abstract Epidemiological, clinical and animal model research has shown that tobacco cigarette smoke (TCS) is a major risk factor for cardiovascular disease (CVD) and metabolic disorders such as hypertension, coronary heart disease, dyslipidemia, and diabetes. Nicotine (NIC), the addicting chemical of TCS, is believed to play a major role in much of the damage induced by TCS. Chronic NIC inhalation has been observed to play a pathogenic role in the induction and progression of CVD. NIC can induce direct coronary spasm and ischemia. It also stimulates both autonomic ganglia and nerve terminals and upregulates numerous vasoconstrictors, inflammatory mediators, cytokines and oxidative markers that play a role in inducing cardiovascular disease. Vascular endothelial dysfunction (VED) and inflammation with enhanced reactive oxygen species (ROS) formation have been detected in many cardiovascular and metabolic disorders associated with NIC exposure. However, fundamental questions remain regarding: 1) the underlying mechanisms by which NIC inhalation (NICI) induces CVD; 2) the correlation between disease and the dose of NIC delivered during exposure; 3) the temporal effects of the dose of NIC delivered on the progression of disease; and 4) possible pharmacological approaches to minimize, arrest or reverse NICI-induced disease. Our research plan will provide answers to these questions using a controlled mouse model with available genetic modifications to explore the disease mechanisms. There are 3 specific aims: 1) To determine the duration and dose intensity required to induce NICI-mediated CVD and VED in a controlled mouse NICI model. The effects of NIC on heart rate, blood pressure, heart structure and function, vascular reactivity, endothelial nitric oxide (NO) production that is critical for normal vascular function, endothelial NO synthase (eNOS), leukocyte activation and ROS formation will be determined. 2) With the doses and duration of NICI that produce CVD and VED, the molecular mechanisms involved will be elucidated. The basis for the alterations in NO production will be determined by investigating the effects of NICI on eNOS levels and functional state, as modulated by its critical cofactor tetrahydrobiopterin (BH4), eNOS redox modification by S-glutathionylation, and its oxidative degradation. The role of leukocyte or tissue NADPH oxidase will be determined with superoxide production from this enzyme measured along with the expression and cellular localization of its subunits. Mice will be studied with knockout of critical NADPH oxidase subunits to assess the role of this critical enzyme in CVD and VED. Superoxide dismutase (SOD) over-expressing transgenic mice will be used to assess the role of superoxide. 3) With the knowledge obtained on the mechanism of NIC-induced CVD, we will evaluate interventions aimed at preventing or ameliorating NICI-induced CVD and VED. This research will provide important insights toward understanding the process and mechanisms of NIC inhalation-induced CVD and identify approaches to minimize or reverse it.